Plant pathogens cause hundreds of millions of dollars in damage to crops in the United States annually and cause significantly more damage worldwide. Traditional plant breeding techniques have developed some plants that resist specific pathogens, but these techniques are limited to genetic transfer within breeding species and can be plagued with the difficulty of introducing non-agronomic traits that are linked to pathogen resistance. Furthermore, traditional breeding has focused on resistance to specific pathogens rather than general, or systemic, resistance to a wide spectrum of pathogens.
One of the most important crop plants in the world is rice. Little is currently known about the mechanisms by which rice resists pathogens. Therefore, an important goal in agriculture is to identify genetic components that enable plants in general, and rice in particular, to resist pathogens, thereby allowing for the development of systemically resistant plants through biotechnology.
Systemic acquired resistance (SAR) is a general plant resistance response that can be induced during a local infection by an avirulent pathogen. While early studies of SAR were conducted using tobacco mosaic virus (TMV) and its Solanaceous hosts (see, e.g., Ross, A. F. Virology 14: 340–358 (1961)), SAR has been demonstrated in many plant species and shown to be effective against not only viruses, but also bacterial and fungal pathogens (see, e.g., Kuc, J. Bioscience 32:854–860 (1982) and Ryals, et al. Plant Cell 8:1809–1819 (1996)). A necessary signal for SAR induction is salicylic acid (SA); plants that fail to accumulate SA due to the expression of an SA-oxidizing enzyme salicylate hydroxylase are impaired in SAR (Gaffney, T., et al. Science 261:754–756 (1993)). Conversely, an elevation in the endogenous level of SA or exogenous application of SA or its synthetic analogs, such as 2,6-dichloroisonicotinic acid (INA), not only results in an enhanced, broad-spectrum resistance but also stimulates concerted expression of a battery of genes known as pathogenesis-related (PR) genes (see, e.g., Malamy, J., et al. Science 250:1002–1004 (1990); Métraux, J.-P., et al. Science 250:1004–1006 (1990); Rasmussen, J. B., et al. Plant Physiol 97:1342–1347 (1991); Yalpani, N., et al. Plant Cell 3:809–818 (1991); White, R. F. Virology 99:410–412 (1979); Métraux, J.-P., et al. (1991) In Advances in Molecular Genetics of Plant-Microbe Interactions, eds. Hennecke, H. & Verma, D. P. S. (Kluwer Academic, Dordrechet, The Netherlands), Vol. 1, pp. 432–439; Ward et al. Plant Cell 3:1085–1094 (1991); and Uknes et al. Plant Cell 4:645–656 (1992)). PR genes may play direct roles in conferring resistance because their expression coincides with the onset of SAR and some of the PR genes encode enzymes with antimicrobial activities (see, e.g., Ward et al. Plant Cell 3:1085–1094 (1991); and Uknes et al. Plant Cell 4:645–656 (1992)). Therefore, understanding the regulation of PR gene expression has been a focal point of research in plant disease resistance.
The Arabidopsis gene NPR1 (Cao et al., Plant Cell 88(1):57–63 (1997) has been shown to be a key component of SA-regulated PR gene expression and disease resistance because npr1 mutants fail to express PR1, PR2, and PR5 and display enhanced susceptibility to infection even after treatment with SA or INA. Furthermore, transgenic plants overexpressing NPR1 display a more dramatic induction of PR genes during an infection and show complete resistance to Pseudomonas syringae pv. maculicola 4326 and Peronospora parasitica Noco, two very different pathogens that are virulent on wild-type A. thaliana plants (Cao, H., et al. Proc. Natl. Acad. Sci. USA 95:6531–6536 (1998)).
NPR1 contains at least four ankyrin repeats, which are found in proteins with very diverse biological functions and are involved in protein—protein interactions (Bork, P. (1993) Proteins: Structure, Function, and Genetics 17, 363–374. Michaely, P., and Bennet, V. (1992) Trends in Cell Biology 2:127–129.). The functional importance of the ankyrin repeat domain has been demonstrated by mutations found in the npr1-1 and the nim1-2 alleles where the highly conserved histidine residues in the third and the second ankyrin repeats, respectively, are changed to a tyrosine. Because these conserved histidine residues are involved in the formation of hydrogen bonds which are crucial in stabilizing the three dimensional structure of the ankyrin-repeat domain (Gorina, S. & Pavletich, N. P. Science 274, 1001–1005 (1996)), npr1-1 and nim1-2 mutations may cause disruption in the local structure within the ankyrin-repeat domain and abolish its ability to interact with other proteins. These data suggest that NPR1 may exert its regulatory function by interacting with other proteins.
bZIP proteins are one class of proteins that interact with NPR1 in a yeast two-hybrid system. bZIP proteins are transcription factors that have highly conserved DNA binding domains. Although functions have been postulated for some plant bZIP gene products (see, e.g., Kawata, T., et al. Nucleic Acids Res. 20, 1141 (1992); Xiang, C., et al. Plant Mol. Biol. 34, 403–415 (1997); Zhang, B., et al. Plant J. 4, 711–716 (1993); Schindler, U., et al., A. R. Plant Cell 4, 1309–1319 (1992); Miao, Z. H., et al. Plant Mol. Biol. 25, 1–11 (1994); and Lam, E. & Lam, Y. K.-P. Nucleic Acids Res. 23, 3778–3785 (1995); Foley et al., Plant J. 3(5):669–79 (1993); Fromm, et al, Mol. Gen. Genet. 229:181–88 (1991); 1998 review, Schwechheimer and Bevan, Trends in Plant Science, 3:378 (1998); and Katagiri et al., Nature 340:727–30 (1989)), little is known about the regulation of bZIP gene products and there are no reports of their interaction with proteins associated with plant disease resistance, other than NPR1.
In spite of recent research of the genetic control of plant resistance to pathogens, little progress has been reported in the identification and analysis of gene products interacting with key regulators of pathogen resistance such as NPR1. Furthermore, most research has been carried out in model plant systems such as Arabidopsis. Little research has been performed on crop plants such as rice. Identification and characterization of rice NPR1 orthologs as well as rice gene products that interact with NPR1 or bZIP gene products would allow for the genetic engineering of plants with a variety of desirable traits. The present invention addresses these and other needs.